- Email: [email protected]
Identification of peptides in functional Scamorza ovine milk cheese
J. Dairy Sci. 98:1–5 http://dx.doi.org/10.3168/jds.2015-9844 © American Dairy Science Association®, 2015.
Identification of peptides in functional Scamorza ovine milk cheese M. Albenzio,1 A. Santillo, R. Marino, A. Della Malva, M. Caroprese, and A. Sevi
Department of the Sciences of Agriculture, Food and Environment (SAFE), University of Foggia, Via Napoli, 25, 71100 Foggia, Italy
ABSTRACT
catalyzed coagulation, and heating and stretching of the curd mass. Albenzio et al. (2013a,b) developed a production protocol and evaluated the characteristics of Scamorza ovine cheese containing probiotics and verified that Bifidobacterium longum, Bifidobacterium lactis, and Lactobacillus acidophilus could survive the heat stress during the stretching phase and persist at high levels in the cheese matrix, influencing the proteolytic, lipolytic, and sensorial properties of the cheese. Milk proteins are currently the main source of a range of biologically active peptides, which have gained special interest because they may influence numerous physiological responses in the organism. Many bioactivities in milk are encrypted within the primary structure of milk proteins, requiring proteolysis for their release from precursors (Gobbetti et al., 2004). In Manchego ovine cheese ripened for 8 mo, several peptides with angiotensin-converting enzyme (ACE)inhibitory activity were identified (Gómez-Ruiz et al., 2004), and antibacterial peptides were obtained in Pecorino Romano ovine cheese (Rizzello et al., 2005). Peptide formation is associated with cheese ripening, thus biologically active peptides can be ingested as naturally occurring components of food (Gómez-Ruiz et al., 2002). The present study was undertaken to evaluate the peptide profile of pasta filata Scamorza ovine cheeses ripened for 15 d and containing either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus.
Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. Key words: pasta filata, ovine cheese, probiotic, peptide INTRODUCTION
Today, many cheeses are available on the market, varying in composition, texture, appearance, and taste. Cheese is a nutritious food, being a rich source of essential nutrients such as fat, fatty acids, proteins, peptides, amino acids, vitamins, and minerals; thus, it can play an essential role in meeting nutritional requirements. Furthermore, cheese represents a good choice as a probiotic food carrier, showing potential advantages for human health over other dairy fermented products; however, the development of probiotic cheese also represents a technological challenge (Castro et al., 2015). Pasta filata cheese varieties are mainly produced from cow and buffalo milks; the principal phases of pasta filata cheese production are acid development, rennet-
MATERIALS AND METHODS
Scamorza cheese production was performed according to the protocol reported in Albenzio et al. (2013a). Briefly, ovine milk was thermized and inoculated with 1% commercial starter (Lyofast, ST044, Sacco, Como, Italy) and with 2% heat-adapted cells of Lactobacillus acidophilus or a mix of Bifidobacterium longum and Bifidobacterium lactis. Lyophilized cultures of Lactobacillus acidophilus, Bifidobacterium longum, and Bifidobacterium lactis were supplied by Mediterranea Biotecnologie S.r.l. (Termoli, Italy). After the milk was cooled, liquid rennet (Chr. Hansen S.p.A., Parma,
Received May 21, 2015. Accepted June 15, 2015. 1 Corresponding author: [email protected]
1
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ALBENZIO ET AL.
Italy) was added, and the curd reached a final pH value of 4.9; then, the curd was stretched mechanically in hot water and molded into a pear shape. Scamorza cheese was brine salted and ripened for 15 d. Cheeses were designated S-CO for control Scamorza cheese without probiotics added, S-BB for Scamorza cheese made using a mix of B. longum and B. lactis, and S-LA for Scamorza cheese made using L. acidophilus as the probiotic strain. Cheeses were analyzed at 15 d of ripening. The pH 4.6-soluble N fraction was obtained according to Kuchroo and Fox (1982). The peptide profiles of the pH 4.6-soluble N fractions were determined by reverse-phase (RP)-HPLC by using the Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA). The column used was a Zorbax 300 SBC18 (250 mm × 4.6 mm × 5 μm; Agilent Technologies). The mobile phase comprised water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid, and the solvent flow rate was 1 mL/min. The eluate was monitored at 220 nm; all solvents were of chromatography grade (Mallinckrodt Baker Inc., Phillipsburg, NJ). Peptides eluting between 1 and 20 min were selected and collected using a fraction collector automatic sampler (G1364C, Agilent Technologies) from each Scamorza cheese sample for subsequent identification. Peptide aliquots were analyzed using a 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-Tof) liquid chromatography mass spectrometry (LC/MS) system (Agilent Technologies) equipped with a 1200 HPLC System and chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed on a 40-nL enrichment column (Agilent Technologies chip), with 0.1% formic acid in 2% acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at a flow rate of 400 nL/min, with a linear gradient of eluent B (0.1% formic acid in 95% acetonitrile) in A (0.1% formic acid in 2% acetonitrile) from 7 to 80% in 50 min. Peptide analysis was performed using data-dependent acquisition of one MS scan (mass range from 300 to 1,800 m/z) followed by a tandem MS (MS/MS) scan of the 5 most abundant ions in each MS scan. The MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50,000 counts. Double and triple charged ions were preferably isolated and fragmented over single charged ions. The acquired MS/MS spectra were transformed in mzData (.XML) format and used for protein identification with a licensed version of MASCOT software (version 2.4.0; www.matrixscience.com). Raw data from nano LC-MS/ MS analysis were used to query the National Center Journal of Dairy Science Vol. 98 No. 12, 2015
for Biotechnology Information nonredundant sequence database (NCBInr database, 20121120; 21,582,400 sequences; 7,401,135,489 residues). The MASCOT search parameters were: trypsin as enzyme; 3 as allowed number of missed cleavage; carboamidomethyl as fixed modification; oxidation of methionine; phosphorylation of serine/threonine/tyrosine; pyro-Glu N-term Q as variable modifications; 10 ppm MS tolerance and 0.6 Da MS/MS tolerance; peptide charge from +2 to +3. The eptide score threshold provided from MASCOT software to evaluate quality of matches for MS/MS data was 25. Spectra with MASCOT score of <25 having low quality were rejected. RESULTS AND DISCUSSION
The RP-HPLC chromatograms of the water-soluble N fraction of Scamorza cheeses at 15 d of ripening are shown in Figure 1. The percentage of pH 4.6-soluble N fraction was highest (P < 0.01) in S-BB, reaching a value of 0.93 ± 0.02% compared with 0.74 ± 0.02% and 0.79 ± 0.02% in S-CO and S-LA, respectively, at 15 d of ripening. The RP-HPLC was focused on early-eluting peaks characterized by high hydrophobicity with a retention time lower than 20 min because this zone showed the major differences among peptides profiles of the Scamorza cheeses in terms of number of peaks and area under the curve. The sum of peak area shown in the chromatograms was 26,497 ± 650 arbitrary units (AU) for S-BB, 24,777 ± 680 AU for S-LA, and 8,780 ± 750 AU for S-CO. In the present study, Scamorza cheeses containing probiotics displayed a profile with higher total area than control cheese, confirming the major effect of probiotics on secondary proteolysis, with the liberation of low-molecular-weight peptides collected in the soluble N fraction of Scamorza cheese after 15 d of ripening (Albenzio et al., 2013a). Within functional Scamorza cheeses, S-BB cheese showed the highest total area, confirming the greater ability of the selected bifidobacteria to control the proteolytic process, as previously observed in Pecorino ovine cheese (Santillo et al., 2009; Albenzio et al., 2010). A qualitative evaluation of the RP-HPLC profiles was performed: 2 peaks were chosen for each cheese type based on major peak area and comparable retention time among Scamorza cheeses. In addition, an additional peak was selected and identified for S-BB cheese because it was unique to the RP-HPLC profile of that Scamorza cheese. Peptides identified in the fraction collected by RPHPLC from the pH 4.6-soluble N fraction of Scamorza cheese at 15 d of ripening are reported in Table 1. The selected peak 1 in S-BB cheese showed co-elution of
FUNCTIONAL SCAMORZA CHEESE
3
Figure 1. Reverse phase-HPLC chromatograms of the water-soluble nitrogen fraction of Scamorza cheeses at 15 d of ripening and area and retention time of identified peaks. S-CO = control Scamorza cheese; S-BB = Scamorza cheese containing the mix of Bifidobacterium longum BL-46 and Bifidobacterium lactis BB-12; S-LA = Scamorza cheese containing Lactobacillus acidophilus LA-5.
2 fragments deriving from β-CN and αS1-CN, respectively. The identified peptides shared structural homology with previously described bioactive peptides in ovine milk and dairy products. In particular, the residue of peptides GPFPI and ENLLRF have been reported to exert ACE-inhibitory activity (Minervini et al. 2003; Meira et al., 2012); RPKHPIK has been found in Cheddar cheese (Ong et al., 2007), in Festivo cheese (Ryhänen et al., 2001), and in Iberian ovine cheese subjected to a hydrolysis process that simulates gastric digestion and is reported to have antimicrobial activity (Recio, 2011). The peptide RPKHPIKHQ was found in a water-soluble peptide preparation isolated from Gouda ripened for 8 mo and found to have potent antihypertensive activity as tested in spontaneously hypertensive rats (Saito et al., 2000). Furthermore, the
fragment 1–23 of αS1-CN known as isracidin originates from the proteolytic activity of chymosin and exerts antimicrobial activity on several microorganisms (Gobbetti et al., 2004). The sequence PQEVLNENLLRF was referenced by Minkiewicz et al. (2000) as an immunomodulating and antimicrobial peptide sequence in the primary structure of αS1-CN released by chymosin activity. Peak 2 was identified as a fragment deriving from β-galactosidase associated with B. longum activity. Peak 3 was the extra peak identified only in S-BB and consisted of 3 peptide fragments of different origin: one fragment derived from κ-CN hydrolysis, the second was of αS1-CN origin, and the third co-eluting fragment was identified as a sequence of endonuclease ascribed to B. longum. Peaks 1 and 2 of S-LA Scamorza cheese were identified as enzymes from Lactobacillus acidophiJournal of Dairy Science Vol. 98 No. 12, 2015
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Table 1. Peptides identified in the fraction collected by reverse phase-HPLC of the soluble nitrogen fractions of Scamorza cheeses1 Cheese and fraction S-BB Peak 1 Peak 2 Peak 3
S-LA Peak 1 Peak 2
Protein fragment
Origin
Sequence
Observed mass
Sequence coverage, %
β-CN αS1-CN β-galactosidase Bifidobacterium longum κ-CN αS1-CN Endonuclease B. longum
f210–220 f1–23 f462–471
VLGPVRGPFPI RPKHPIKHQGLPQEVLNENLLRF KAGAESGGMEMS
1,150.7 2,221.5 1,153.47
100 91 70
f78–90 f1–6 f15–28
SAEPTVHSTPTTE RPKHPI ETHLEAQPTVASAQ
1,355.62 746.45 1,480.71
100 100 64
Glycerophosphodiester phosphodiesterase family protein Lactobacillus acidophilus ATP-dependent metallo peptidase HflB Lactobacillus acidophilus
f79–97
LALLFSILVLFIMPM
1,720
f512–518
RGGGAGEEE
954.4
42 60
1 S-BB = Scamorza cheese containing a mix of Bifidobacterium longum BL-46 and Bifidobacterium lactis BB-12; S-LA = Scamorza cheese containing Lactobacillus acidophilus LA-5.
lus. The peptides isolated from the 2 peaks of the pH 4.6-soluble N fraction of control Scamorza cheese were not attributed to specific peptide sequences and therefore were not included in Table1. Identified peaks were partly ascribed to casein hydrolysis and partly to enzymes associated with the probiotics added into Scamorza cheeses during cheesemaking. It is worth noting that among the identified peaks, only fragments in S-BB cheese yielded peptides referenced as bioactive sequences. In particular, B. longum and B. lactis showed greater adaptation than L. acidophilus to the heat-stress conditions of the protocol for Scamorza cheese production; indeed, bifidobacteria and L. acidophilus showed cell loads of 9.09 ± 0.04 log10 cfu/g and 7.55 ± 0.07 log10 cfu/g, respectively, at 15 d of ripening (Albenzio et al., 2013a). The higher bifidobacteria cell load recovered in Scamorza ovine cheese led to higher casein hydrolysis and a subsequent higher level of pH 4.6-soluble N fraction, with the liberation of peptides from milk protein that have potential functionality. CONCLUSIONS
In functional Scamorza cheeses containing either a mix of B. longum and B. lactis or L. acidophilus, specific peptides deriving from microbial enzymes may be regarded as tracing fragments and represent a tool to verify the presence and activity of probiotic cultures in cheese. Scamorza ovine cheese ripened for 15 d exhibited the presence of bioactive sequences previously reported in long-ripened cheeses, demonstrating the ability of probiotics to control the proteolytic process and generate peptides in the cheese matrix that could be delivered upon cheese consumption. Journal of Dairy Science Vol. 98 No. 12, 2015
ACKNOWLEDGMENTS
The present work is part of the Research Program titled “Innovative strategies for quality and safety in ovine production” financially supported by the Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF; Rome, Italy). REFERENCES Albenzio, M., A. Santillo, M. Caroprese, A. Braghieri, A. Sevi, and F. Napolitano. 2013a. Composition and sensory profiling of probiotic Scamorza ewe’s milk cheese. J. Dairy Sci. 96:2792–2800. Albenzio, M., A. Santillo, M. Caroprese, R. Marino, A. Trani, and M. Faccia. 2010. Biochemical patterns in ovine cheese: Influence of probiotic strains. J. Dairy Sci. 93:3487–3496. Albenzio, M., A. Santillo, M. Caroprese, D. Ruggieri, F. Napolitano, and A. Sevi. 2013b. Physicochemical properties of Scamorza ovine cheese manufactured with different probiotic cultures. J. Dairy Sci. 96:2781–2791. Castro, J. M., M. E. Tornadijo, J. M. Fresno, and H. Sandoval. 2015. Biocheese: A food probiotic carrier. BioMed Res. Int. 2015:723056. http://dx.doi.org/10.1155/2015/723056. Gobbetti, M., F. Minervini, and C. Rizzello. 2004. Angiotensin I-converting-enzyme-inhibitory and antimicrobial bioactive peptides. Int. J. Dairy Technol. 57:173–188. Gómez-Ruiz, J. A., M. Ramos, and I. Recio. 2002. Angiotensin-converting enzyme-inhibitory peptides in Manchego cheeses manufactured with different starter cultures. Int. Dairy J. 12:697–706. Gómez-Ruiz, J. A., M. Ramos, and I. Recio. 2004. Angiotensin-converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. Int. Dairy J. 14:1075–1080. Kuchroo, C. N., and P. F. Fox. 1982. Soluble nitrogen in Cheddar cheese: Comparison of extraction procedures. Milchwissenschaft 37:331–335. Meira, S. M. M. M., D. J. Daroit, V. E. Helfer, A. P. Folmer Corrêa, J. Segalin, S. Carro, and A. Brandelli. 2012. Bioactive peptides in water-soluble extracts of ovine cheeses from Southern Brazil and Uruguay. Food Res. Int. 48:322–329. Minervini, F., F. Algaron, C. G. Rizzello, P. F. Fox, V. Monnet, and M. Gobbetti. 2003. Angiotensin I-converting-enzyme-inhibitory
FUNCTIONAL SCAMORZA CHEESE
and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolysed caseins of milk of six species. Appl. Environ. Microbiol. 69:5297–5305. Minkiewicz, P., C. J. Slangen, J. Dziuba, S. Visser, and H. Mioduszewska. 2000. Identification of peptides obtained via hydrolysis of bovine casein using HPLC and mass spectrometry. Milchwissenschaft 55:14–17. Ong, L., A. Henriksson, and N. P. Shah. 2007. Chemical analysis and sensory evaluation of Cheddar cheese produced with Lactobacillus acidophilus, Lb. casei, Lb. paracasei or Bifidobacterium sp. Int. Dairy J. 17:937–945. Recio, I. 2011. Health and nutritional aspects of cheese with a focus on the bioactive peptides. Proc. 8th Cheese Symposium 2011, Moorepark, Cork, Ireland. Dairy Sci. Technol. 91(Special Issue):31.
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Rizzello, C. G., I. Losito, M. Gobbetti, T. Carbonara, M. D. De Bari, and P. Zambonin. 2005. Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties. J. Dairy Sci. 88:2348–2360. Ryhänen, E. L., A. Pihlanto-Leppälä, and E. Pahkala. 2001. A new type ripened low-fat cheese with bioactive properties. Int. Dairy J. 11:441–447. Saito, T., T. Nakamura, H. Kitazawa, Y. Kawai, and T. Itoh. 2000. Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese. J. Dairy Sci. 83:1434–1440. Santillo, A., M. Albenzio, M. Quinto, M. Caroprese, R. Marino, and A. Sevi. 2009. Probiotic in lamb rennet paste enhances rennet lipolytic activity, and CLA and linoleic acid content in Pecorino cheese. J. Dairy Sci. 92:1330–1337.
Journal of Dairy Science Vol. 98 No. 12, 2015
Identification of peptides in functional Scamorza ovine milk cheese M. Albenzio,1 A. Santillo, R. Marino, A. Della Malva, M. Caroprese, and A. Sevi
Department of the Sciences of Agriculture, Food and Environment (SAFE), University of Foggia, Via Napoli, 25, 71100 Foggia, Italy
ABSTRACT
catalyzed coagulation, and heating and stretching of the curd mass. Albenzio et al. (2013a,b) developed a production protocol and evaluated the characteristics of Scamorza ovine cheese containing probiotics and verified that Bifidobacterium longum, Bifidobacterium lactis, and Lactobacillus acidophilus could survive the heat stress during the stretching phase and persist at high levels in the cheese matrix, influencing the proteolytic, lipolytic, and sensorial properties of the cheese. Milk proteins are currently the main source of a range of biologically active peptides, which have gained special interest because they may influence numerous physiological responses in the organism. Many bioactivities in milk are encrypted within the primary structure of milk proteins, requiring proteolysis for their release from precursors (Gobbetti et al., 2004). In Manchego ovine cheese ripened for 8 mo, several peptides with angiotensin-converting enzyme (ACE)inhibitory activity were identified (Gómez-Ruiz et al., 2004), and antibacterial peptides were obtained in Pecorino Romano ovine cheese (Rizzello et al., 2005). Peptide formation is associated with cheese ripening, thus biologically active peptides can be ingested as naturally occurring components of food (Gómez-Ruiz et al., 2002). The present study was undertaken to evaluate the peptide profile of pasta filata Scamorza ovine cheeses ripened for 15 d and containing either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus.
Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. Key words: pasta filata, ovine cheese, probiotic, peptide INTRODUCTION
Today, many cheeses are available on the market, varying in composition, texture, appearance, and taste. Cheese is a nutritious food, being a rich source of essential nutrients such as fat, fatty acids, proteins, peptides, amino acids, vitamins, and minerals; thus, it can play an essential role in meeting nutritional requirements. Furthermore, cheese represents a good choice as a probiotic food carrier, showing potential advantages for human health over other dairy fermented products; however, the development of probiotic cheese also represents a technological challenge (Castro et al., 2015). Pasta filata cheese varieties are mainly produced from cow and buffalo milks; the principal phases of pasta filata cheese production are acid development, rennet-
MATERIALS AND METHODS
Scamorza cheese production was performed according to the protocol reported in Albenzio et al. (2013a). Briefly, ovine milk was thermized and inoculated with 1% commercial starter (Lyofast, ST044, Sacco, Como, Italy) and with 2% heat-adapted cells of Lactobacillus acidophilus or a mix of Bifidobacterium longum and Bifidobacterium lactis. Lyophilized cultures of Lactobacillus acidophilus, Bifidobacterium longum, and Bifidobacterium lactis were supplied by Mediterranea Biotecnologie S.r.l. (Termoli, Italy). After the milk was cooled, liquid rennet (Chr. Hansen S.p.A., Parma,
Received May 21, 2015. Accepted June 15, 2015. 1 Corresponding author: [email protected]
1
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ALBENZIO ET AL.
Italy) was added, and the curd reached a final pH value of 4.9; then, the curd was stretched mechanically in hot water and molded into a pear shape. Scamorza cheese was brine salted and ripened for 15 d. Cheeses were designated S-CO for control Scamorza cheese without probiotics added, S-BB for Scamorza cheese made using a mix of B. longum and B. lactis, and S-LA for Scamorza cheese made using L. acidophilus as the probiotic strain. Cheeses were analyzed at 15 d of ripening. The pH 4.6-soluble N fraction was obtained according to Kuchroo and Fox (1982). The peptide profiles of the pH 4.6-soluble N fractions were determined by reverse-phase (RP)-HPLC by using the Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA). The column used was a Zorbax 300 SBC18 (250 mm × 4.6 mm × 5 μm; Agilent Technologies). The mobile phase comprised water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid, and the solvent flow rate was 1 mL/min. The eluate was monitored at 220 nm; all solvents were of chromatography grade (Mallinckrodt Baker Inc., Phillipsburg, NJ). Peptides eluting between 1 and 20 min were selected and collected using a fraction collector automatic sampler (G1364C, Agilent Technologies) from each Scamorza cheese sample for subsequent identification. Peptide aliquots were analyzed using a 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-Tof) liquid chromatography mass spectrometry (LC/MS) system (Agilent Technologies) equipped with a 1200 HPLC System and chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed on a 40-nL enrichment column (Agilent Technologies chip), with 0.1% formic acid in 2% acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at a flow rate of 400 nL/min, with a linear gradient of eluent B (0.1% formic acid in 95% acetonitrile) in A (0.1% formic acid in 2% acetonitrile) from 7 to 80% in 50 min. Peptide analysis was performed using data-dependent acquisition of one MS scan (mass range from 300 to 1,800 m/z) followed by a tandem MS (MS/MS) scan of the 5 most abundant ions in each MS scan. The MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50,000 counts. Double and triple charged ions were preferably isolated and fragmented over single charged ions. The acquired MS/MS spectra were transformed in mzData (.XML) format and used for protein identification with a licensed version of MASCOT software (version 2.4.0; www.matrixscience.com). Raw data from nano LC-MS/ MS analysis were used to query the National Center Journal of Dairy Science Vol. 98 No. 12, 2015
for Biotechnology Information nonredundant sequence database (NCBInr database, 20121120; 21,582,400 sequences; 7,401,135,489 residues). The MASCOT search parameters were: trypsin as enzyme; 3 as allowed number of missed cleavage; carboamidomethyl as fixed modification; oxidation of methionine; phosphorylation of serine/threonine/tyrosine; pyro-Glu N-term Q as variable modifications; 10 ppm MS tolerance and 0.6 Da MS/MS tolerance; peptide charge from +2 to +3. The eptide score threshold provided from MASCOT software to evaluate quality of matches for MS/MS data was 25. Spectra with MASCOT score of <25 having low quality were rejected. RESULTS AND DISCUSSION
The RP-HPLC chromatograms of the water-soluble N fraction of Scamorza cheeses at 15 d of ripening are shown in Figure 1. The percentage of pH 4.6-soluble N fraction was highest (P < 0.01) in S-BB, reaching a value of 0.93 ± 0.02% compared with 0.74 ± 0.02% and 0.79 ± 0.02% in S-CO and S-LA, respectively, at 15 d of ripening. The RP-HPLC was focused on early-eluting peaks characterized by high hydrophobicity with a retention time lower than 20 min because this zone showed the major differences among peptides profiles of the Scamorza cheeses in terms of number of peaks and area under the curve. The sum of peak area shown in the chromatograms was 26,497 ± 650 arbitrary units (AU) for S-BB, 24,777 ± 680 AU for S-LA, and 8,780 ± 750 AU for S-CO. In the present study, Scamorza cheeses containing probiotics displayed a profile with higher total area than control cheese, confirming the major effect of probiotics on secondary proteolysis, with the liberation of low-molecular-weight peptides collected in the soluble N fraction of Scamorza cheese after 15 d of ripening (Albenzio et al., 2013a). Within functional Scamorza cheeses, S-BB cheese showed the highest total area, confirming the greater ability of the selected bifidobacteria to control the proteolytic process, as previously observed in Pecorino ovine cheese (Santillo et al., 2009; Albenzio et al., 2010). A qualitative evaluation of the RP-HPLC profiles was performed: 2 peaks were chosen for each cheese type based on major peak area and comparable retention time among Scamorza cheeses. In addition, an additional peak was selected and identified for S-BB cheese because it was unique to the RP-HPLC profile of that Scamorza cheese. Peptides identified in the fraction collected by RPHPLC from the pH 4.6-soluble N fraction of Scamorza cheese at 15 d of ripening are reported in Table 1. The selected peak 1 in S-BB cheese showed co-elution of
FUNCTIONAL SCAMORZA CHEESE
3
Figure 1. Reverse phase-HPLC chromatograms of the water-soluble nitrogen fraction of Scamorza cheeses at 15 d of ripening and area and retention time of identified peaks. S-CO = control Scamorza cheese; S-BB = Scamorza cheese containing the mix of Bifidobacterium longum BL-46 and Bifidobacterium lactis BB-12; S-LA = Scamorza cheese containing Lactobacillus acidophilus LA-5.
2 fragments deriving from β-CN and αS1-CN, respectively. The identified peptides shared structural homology with previously described bioactive peptides in ovine milk and dairy products. In particular, the residue of peptides GPFPI and ENLLRF have been reported to exert ACE-inhibitory activity (Minervini et al. 2003; Meira et al., 2012); RPKHPIK has been found in Cheddar cheese (Ong et al., 2007), in Festivo cheese (Ryhänen et al., 2001), and in Iberian ovine cheese subjected to a hydrolysis process that simulates gastric digestion and is reported to have antimicrobial activity (Recio, 2011). The peptide RPKHPIKHQ was found in a water-soluble peptide preparation isolated from Gouda ripened for 8 mo and found to have potent antihypertensive activity as tested in spontaneously hypertensive rats (Saito et al., 2000). Furthermore, the
fragment 1–23 of αS1-CN known as isracidin originates from the proteolytic activity of chymosin and exerts antimicrobial activity on several microorganisms (Gobbetti et al., 2004). The sequence PQEVLNENLLRF was referenced by Minkiewicz et al. (2000) as an immunomodulating and antimicrobial peptide sequence in the primary structure of αS1-CN released by chymosin activity. Peak 2 was identified as a fragment deriving from β-galactosidase associated with B. longum activity. Peak 3 was the extra peak identified only in S-BB and consisted of 3 peptide fragments of different origin: one fragment derived from κ-CN hydrolysis, the second was of αS1-CN origin, and the third co-eluting fragment was identified as a sequence of endonuclease ascribed to B. longum. Peaks 1 and 2 of S-LA Scamorza cheese were identified as enzymes from Lactobacillus acidophiJournal of Dairy Science Vol. 98 No. 12, 2015
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Table 1. Peptides identified in the fraction collected by reverse phase-HPLC of the soluble nitrogen fractions of Scamorza cheeses1 Cheese and fraction S-BB Peak 1 Peak 2 Peak 3
S-LA Peak 1 Peak 2
Protein fragment
Origin
Sequence
Observed mass
Sequence coverage, %
β-CN αS1-CN β-galactosidase Bifidobacterium longum κ-CN αS1-CN Endonuclease B. longum
f210–220 f1–23 f462–471
VLGPVRGPFPI RPKHPIKHQGLPQEVLNENLLRF KAGAESGGMEMS
1,150.7 2,221.5 1,153.47
100 91 70
f78–90 f1–6 f15–28
SAEPTVHSTPTTE RPKHPI ETHLEAQPTVASAQ
1,355.62 746.45 1,480.71
100 100 64
Glycerophosphodiester phosphodiesterase family protein Lactobacillus acidophilus ATP-dependent metallo peptidase HflB Lactobacillus acidophilus
f79–97
LALLFSILVLFIMPM
1,720
f512–518
RGGGAGEEE
954.4
42 60
1 S-BB = Scamorza cheese containing a mix of Bifidobacterium longum BL-46 and Bifidobacterium lactis BB-12; S-LA = Scamorza cheese containing Lactobacillus acidophilus LA-5.
lus. The peptides isolated from the 2 peaks of the pH 4.6-soluble N fraction of control Scamorza cheese were not attributed to specific peptide sequences and therefore were not included in Table1. Identified peaks were partly ascribed to casein hydrolysis and partly to enzymes associated with the probiotics added into Scamorza cheeses during cheesemaking. It is worth noting that among the identified peaks, only fragments in S-BB cheese yielded peptides referenced as bioactive sequences. In particular, B. longum and B. lactis showed greater adaptation than L. acidophilus to the heat-stress conditions of the protocol for Scamorza cheese production; indeed, bifidobacteria and L. acidophilus showed cell loads of 9.09 ± 0.04 log10 cfu/g and 7.55 ± 0.07 log10 cfu/g, respectively, at 15 d of ripening (Albenzio et al., 2013a). The higher bifidobacteria cell load recovered in Scamorza ovine cheese led to higher casein hydrolysis and a subsequent higher level of pH 4.6-soluble N fraction, with the liberation of peptides from milk protein that have potential functionality. CONCLUSIONS
In functional Scamorza cheeses containing either a mix of B. longum and B. lactis or L. acidophilus, specific peptides deriving from microbial enzymes may be regarded as tracing fragments and represent a tool to verify the presence and activity of probiotic cultures in cheese. Scamorza ovine cheese ripened for 15 d exhibited the presence of bioactive sequences previously reported in long-ripened cheeses, demonstrating the ability of probiotics to control the proteolytic process and generate peptides in the cheese matrix that could be delivered upon cheese consumption. Journal of Dairy Science Vol. 98 No. 12, 2015
ACKNOWLEDGMENTS
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Journal of Dairy Science Vol. 98 No. 12, 2015